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Design, engineering, and characterization of zinc finger nucleases
Abstract
Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.
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“Mani et al. showed that three-finger ZFN was able to specifically cleave CCR5 at the designed site using an in vitro transcription-translation system.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [37]
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Metadata
- Authors
- Mani, M., Kandavelou, K., Dy, F. J., Durai, S., & Chandrasegaran, S.
- Affiliations
- Johns Hopkins University (US)
- Corresponding author
- Chandrasegaran, Srinivasan
- ORCID
- Dy, Fei Jamie
- Venue
- Biochemical and Biophysical Research Communications
- Publisher
- Elsevier BV
- Published
- 2005-09
- Volume
- 335
- Issue
- 2
- Pages
- 447-457
- ISSN
- 0006-291X (ISSN-L)
- OpenAlex
- W1972389078
- Cited by
- 129
- References
- 39
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 6 · 2023: 4 · 2022: 4 · 2021: 5 · 2020: 3 · 2019: 4 · 2018: 4 · 2017: 2 · 2016: 7 · 2015: 7 · 2014: 6 · 2013: 7 · 2012: 8
- FWCI
- 3.36
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemical and Biophysical Research Communications (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM053923, GM 53923)
- National Institute of Allergy and Infectious Diseases (P30AI042855)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- zinc finger nucleases, gene targeting, genome editing, directed mutagenesis, sequence-specific cleavage, DNA double-strand breaks, tyrosinase, CFTR, CCR5, DMPK
- MeSH terms
- Amino Acid Sequence, Animals, Base Sequence, DNA, DNA Damage, Endoribonucleases, Genetic Techniques, Humans, Molecular Sequence Data, Plasmids, Rabbits, Recombination, Genetic, Transcription, Genetic, Protein Biosynthesis, Blotting, Western, Protein Engineering, Genome, Human, Polymerase Chain Reaction, Mutagenesis, Site-Directed, Zinc Fingers, Genome, Protein Serine-Threonine Kinases, Protein Structure, Tertiary, Genome, Plant, Receptors, CCR5, Amino Acid Motifs, Mice, Myotonin-Protein Kinase
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